The study of our most distant animal relatives through the use of phylogenetic and comparative genomic approaches has significantly advanced our understanding of the relationship between genomic and morphological complexity, the evolution of multicellularity, and the emergence of novel cell types. These findings are leading to the establishment of new model organisms that have the potential to inform important questions in human biology and human health, laying the groundwork for translational studies focused on specific human diseases. The cnidarians, organisms unified in a single phylum based on their use of cnidocytes to capture prey and for defense from predators, occupy a key phylogenetic position as the sister group to the bilaterians. Previous phylogenomic analyses performed by our group have revealed that the genomes of cnidarians encode more homologs to human disease genes than do classic invertebrate models (1), strongly positioning the cnidarians as powerful model systems for the study of biological processes such as pluripotency, regeneration, lineage commitment, and allorecognition. Given their experimental tractability, including the ability to perform CRISPR/Cas9-mediated gene knock-in (2), we are actively sequencing and annotating the genomes of two Hydractinia species: H. echinata and H. symbiolongicarpus. What makes these simple organisms particularly well-suited as a model system lies in the fact that they possess a specific type of interstitial cell (or i-cell) that is pluripotent and provides the basis for tissue regeneration, expressing genes whose bilateral homologs are known to be involved in stem cell biology. Hydractinia is also colonial, possessing an allorecognition system that may provide insights into important questions related to host-graft rejection. Using PacBio, Illumina, and Dovetail-based strategies, high-coverage sequencing data indicate an estimated genome size of 774 Mb for H. echinata (84x coverage) and 514 Mb for H. symbiolongicarpus (94x coverage); these genomes are AT-rich (65%) and highly repetitive (47-51%). The N50 for the H. symbiolongicarpus genome exceeds 2.2 MB, making this one of the most contiguous animal genomes sequenced to date. The vast majority of a set of evolutionarily conserved single-copy orthologs can be easily identified in these assemblies, and analyses of these whole-genome sequencing data have already provided important insights into the evolution of chromatin compaction and metazoan neurogenesis. With respect to the highly repetitive nature of these genomes, repetitive DNA has been implicated in chromatin organization, regulation of gene expression, and the maintenance of genome integrity, but large repeats are often not found in reference genomes due to inherent technical challenges related to sequencing and assembly. Despite these challenges, we have been able to determine the consensus sequence and genomic architecture of rDNA repeats in Hydractinia. Compared to human, Hydractinia has four times as many rDNA repeats in its genome and, while the coding sequences for each ribosomal component are similarly organized and roughly the same size, its intergenic spacers (IGSs) are 100 times shorter than those seen in humans. Given their significantly shorter size, we are exploring whether control elements found in human and vertebrate IGSs are located outside the Hydractinia rDNA cluster. If so, the entire rDNA array may be under the control of a single promoter, enabling it to meet the high demand for ribosomes during regeneration. The analysis of these Hydractinia genomes has also revealed a heretofore unappreciated complexity of the mechanisms underlying allorecognition. Previously, it was thought that two genes (named Alr1 and Alr2) found within the allorecognition complex (ARC) controlled the ability of colonies to distinguish self from non-self through potential signal transduction motifs in their extracellular domains. Analysis of our highly contiguous whole-genome sequence data has revealed there are 10 candidate Alr genes located within the 12 Mb allorecognition complex, with fusion assays indicating that either Alr3 or Alr4 is a putative third allodeterminant (manuscript in preparation). The genomic architecture of the ARC is similar to that of mammalian natural killer cell receptors that also exhibit high levels of allelic polymorphism, gene duplication, and copy number variation, suggesting common mechanisms of genomic evolution in both systems. Future work is focused on the extensive sequence diversity seen in the cytoplasmic tails of these Alr proteins and whether recombination in this genomic region has the potential to create novel binding specificities. While sexual reproduction is ubiquitous among eukaryotes, with sex determination strategies varying greatly among different species, little is known about the mechanisms that determine sex in non-bilaterians. In collaboration with our colleagues at the University of Pittsburgh, we were able to identify a 3 Mb genomic region that co-segregates with sex in Hydractinia. The strategy used to identify this region was based on the construction of a linkage map and subsequent analysis for QTLs linked to sex. 15 linkage groups were recovered in both the male and female maps, corresponding to the haploid chromosome number for Hydractinia. These maps were then used to perform quantitative trait locus analysis to identify markers linked to sex. No sex-linked QTLs were identified on the female map, but a single region on one chromosome of the male map was found to be significantly correlated with sex (LOD > 18, p < 0.001). This region includes four SNPs spanning 3 Mb that co-segregated with sex in 84 of the 87 offspring. These results are consistent with a genetic sex determination system in which males are the heterogametic sex (XX/XY; manuscript in preparation). Finally, clonal animals such as Hydractinia do not sequester a germline during embryogenesis, instead producing gametes from adult stem cells that can also contribute to somatic tissues. However, how germ fate is induced in these animals and whether this process is related to bilateral embryonic germline induction remains an open question. Along with our collaborators at the University of Ireland-Galway, we have shown that transcription factor AP2 (Tfap2), a major regulator of mammalian germline induction, acts as a molecular switch that commits i-cells to germ fate in Hydractinia. Tfap2 mutants were shown to lack germ cells, developing only rudimentary gonads, while transplanted allogenic wild-type cells rescued gonad development but not germ cell induction in Tfap2 mutants. Further, forced expression of Tfap2 in i-cells converted them to germ cells ectopically in non-gonadal tissues of embryos and juveniles, but Tfap2 expression produced no discernible phenotype in somatic cells. These data show that Tfap2 acts cell-autonomously and is essential and sufficient to induce germ cell fate in i-cells, also acting non-cell-autonomously downstream of germ cell induction to promote gonad development. Therefore, Tfap2 is a conserved regulator of germ cell commitment across germline-sequestering and germline-non-sequestering animals (manuscript under review). (1) Maxwell, E.K. et al. BMC Evolutionary Biology 14: 212, 2014. (2) Sanders, S.M. et al. BMC Genomics 19: 649, 2018.